Summary Homologous recombination is a high-fidelity DNA repair process that promotes the integrity of the eukaryotic genome. Recombination can be initiated either by double-strand breaks (DSBs) or single-strand gaps, with individual events being resolved to produce a non-crossover or crossover product (NCO or CO, respectively). Because CO events are uniquely associated with chromosome rearrangements, regulation of the CO-NCO outcome is of particular relevance to genome stability. The proposed research will use a transformation-based, gap-repair assay and a chromosomal, HO-based assay as models for mitotic DSB repair in the yeast Saccharomyces cerevisiae. The advantages of these assays are that the position and nature of the recombination-initiating lesion are known, both CO and NCO events can be isolated, and the presence of silent sequence polymorphisms allows the molecular structure of recombination intermediates to be determined. Each of three aims will focus on understanding precisely how defined activities regulate the CO-NCO outcome. One aim will examine the nucleases that resect the 5 strands of the broken ends, thereby generating the 3 tails required to initiate strand invasion. We propose that the extent of resection is linked to recombination pathway choice and NCO-CO outcome, and this will be tested. A second aim will examine the Mph1, Sgs1 and Srs2 helicases, each of which independently promotes the NCO outcome during DSB repair. A third aim will focus on the Holliday junction, the formation and resolution of which is required to generate CO products. These three aims will be accomplished by examining the extents and positions of strand transfer during individual recombination events. A final aim will focus on understanding how the MMR machinery limits recombination between diverged sequences, an activity that is directed primarily towards CO intermediates. Basic knowledge gained from these studies will be relevant to issues of genome stability in high eukaryotes.